Since Jenner successfully vaccinated a patient against smallpox more than 200 years ago, physicians have sought ways to enhance immunity to deal with other diseases. Immunization in several forms has led to the elimination of serious consequences or even deaths from a number of diseases such as poliomyelitis, diphtheria, pertussis, typhoid fever, mumps and rubella.
Enhancement of immunity is a desirable goal in the treatment of patients diagnosed with cancer, immune deficiency syndrome, certain topical and systemic infections, leprosy, tuberculosis, shingles, warts, herpes, malaria, gingivitis, atherosclerosis, and others.
Almost a hundred years ago William Coley tried to enlist the aid of the immune system against cancer. He had some success in treating cancer patients by infecting them with live bacteria. However, the infections caused such serious problems for the patients that he turned to the development of vaccines based on killed bacteria (Old, L. J. (1988), "Tumor Necrosis Factor," Scientific American 258:59-60). These preparations were called Coley's toxins. He achieved only limited success. Soon radiation and chemotherapy supplanted his toxins as the therapy of choice.
However, investigators continued to be fascinated with the possibility of enhancing immunity to destroy tumors. Lewis Thomas and Macfarlane Burnet gave impetus to the movement when they suggested in their Theory of Immune Surveillance that the body routinely must deal with mutations and therefore carries on a constant surveillance for such rogue cells. The recognition that human tumors are immunogenic to the host and therefore do provoke an immune response, albeit inadequate, has caused investigators to hope that immunotherapy for cancer is possible. (Patillo, R. (1974) "Trophoblast Cancers," in Hormones and Cancer, (K. W. McKerns, ed.), Academic Press, New York, p. 384; Prehn, R. T. (1976), "Do Tumors Grow because of the Immune Response of the Host?" Transplantation Reviews 28:34-42; Ellenhorn, J. D. et al. (1988), "In vivo Administration of Anti-CD3 Prevents Malignant Progressor Tumor Growth," Science 242:569-571.
Rare but indisputable spontaneous regressions of tumors, often after an infection, provided still further encouragement. The exquisite specificity of the immune system, its ability to provide resistance to infections and to destroy transplants, gave promise that enhanced immunity would be the ideal treatment for cancer. However, there have been many attempts but with only limited results.
The first breakthrough in understanding immune regulation was provided by the Clonal Selection Theory developed by David Talmage, Niels Jerne and Macfarlane Burnet, proposing that when antigen and lymphocyte receptor match, the lymphocyte is "selected" for activation and proliferation. However, despite the enormous contribution of the concept of clonal selection to the understanding of immune regulation, little progress has been made in finding a way to enhance immunity. The clinician could reduce immune reactions with such agents as cyclosporine or the glucocorticoids, but he could not increase it.
The second important clue came with the discovery of a group of lymphokines and cytokines. Antigen-nonspecific modulatory proteins released primarily, but not exclusively, by immune cells were shown to regulate the magnitude of the immune response. While only antigen binding could select the cell that would expand into a clone, that cell could proliferate only if it also received stimulation from one or more of these proteins. Some of these substances, such as the interferons or interleukin-1 or -2, directly stimulate the immune cells. Others stimulate the bone marrow to produce more leukocytes. (Golde, D. W. et al. (1988), "Hormones that Stimulate the Growth of Blood Cells," Scientific American 259:62-71).
Some act as chemoattractants to macrophages and polymorphonuclear leukocytes, induce receptors on endothelial cells so that circulating leukocytes stick to them (Bevilacquam M. J. et al. (1989), "Endothelial Leukocyte Adhesion Molecule 1: an Inducible Receptor for Neutrophils Related to Complement Regulatory Proteins and Lectins," Science 243:1160) and cause endothelial cells to part so leukocytes can leave the vascular compartment and enter the wound. Some are potent angiogenic factors, stimulating replacement of vessels in injured tissue. Some immobilize phagocytes to prevent their wandering away from the wound. Others activate macrophages to phagocytose debris and pathogens and to produce more factors (e.g., cachectin and interleukin-1). Fibroblasts are stimulated to lay down fibrin barricades to restrict pathogens to the local area. One cytokine, cachectin (Tumor Necrosis Factor-alpha), appears to be the substance responsible for the effects Coley witnessed. (Old, L. J. (1988), "Tumor Necrosis Factor," Scientific American 258:59 60.)
There have been massive efforts to enlist the assistance of these substances to enhance immunity and to provoke an attack on tumors. Some success has been achieved. Interferon-alpha, produced primarily by leukocytes, has been effective in treating low-grade non-Hodgkin's lymphoma, cutaneous T cell lymphoma, chronic myelogenous leukemia, Kaposi's sarcoma and especially hairy cell leukemia. For reasons not fully understood, it seems to have little effect on some patients and no effect on tumors other than those listed above. (Oppenheim, J. J. et al. (1987), in Basic and Clinical Immunology, Sixth ed., (D. P. Stites, et al., eds.), Appleton and Lange, Los Altos, Calif., p. 94.
Efforts to use cachectin to provoke an inflammation that will be destructive of tumors have produced frustratingly inconsistent results and dangerous toxicity. (Old, L. J. (May 1988), "Tumor Necrosis Factor," Scientific American 258:59-60).
Interleukin-2, produced by helper T cells, has been intensively studied for its effect on tumors. In the most successful of these studies, lymphocytes that have infiltrated a tumor are activated in vitro by IL-2. When the cells have proliferated to significant amounts they, with additional IL-2, are injected into the patient. (Rosenberg, S. A. et al. (1986), "A New Approach to the Adoptive Immunotherapy of Cancer with Tumor-infiltrating Lymphocytes," Science 233:1318-1321; Rosenberg, S. A., (1990), "Adoptive Immunotherapy for Cancer," Scientific American 262:62-69; Geiger, J. D. et al. (1993), "Generation of T-Cells Reactive to the Poorly Immunogenic B16-BL6 Melanoma with Efficacy in the Treatment of Spontaneous Metastases," J. Immunother. 13:153-165; Chang, A. E. et al. (1993), "Clinical Observations on Adoptive Immunotherapy with Vaccine-primed T-Lymphocytes Secondarily Sensitized to Tumor In Vitro," Cancer Res. 53:1043-1050). The experiments have been partially successful in treating colon and kidney cancer and malignant melanoma. About 20% of the patients show at least some response. A few have been symptom-free for months. Unfortunately, IL-2 has been shown to be toxic in the amounts most helpful in dealing with the tumor. (Oppenheim, J. J. et al. (1987), in Basic and Clinical Immunology, Sixth ed., (D. P. Stites, et al., eds.), Appleton and Lange, Los Altos, Calif., p. 94; Petska, S. (ed.), (January 1992), "Current Approaches and Obstacles to Immunotherapy," Pharm. Tech., pp. 26-35.) Toxic effects include malaise, fever, nausea or vomiting, diarrhea and anemia. A nontoxic method for stimulating selected lymphocytes in vivo or in vitro is therefore needed.
When a lymphocyte is selected by antigen, the first detectable effect is an efflux of potassium and a compensating influx of sodium, down the electrochemical gradient. Before the lymphocyte can respond to stimulation by cytokines, it must restore intracellular levels of potassium to normal with the sodium/potassium pump (Na.sup.+ K.sup.+ ATPase), an energy-dependent action. A number of investigators have shown that blockage of the Na.sup.+ K.sup.+ ATPase will prevent the cell from activating or proliferating. (See, e.g., Ward, P. A., (1985) "Inflammation," in Immunology III, (J. A. Bellanti, ed.), W. B. Saunders, Co., Philadelphia, Pa., p. 212; Handwerger, B. S. and Douglas, S. D., (1980) "Cell Biology of Blastogenesis," in The Cell Biology of Inflammation, (G. Weissmann, ed.), Elsevier, New York/N. Holland Biomedical Press, Amsterdam, p. 654.) Tumor cells have been reported to become resistant to chemotherapy and it has been suggested that stimulation of the sodium-potassium ion pump might reverse this drug resistance. Lawrence, T. S. and Davis, M. A. (1990), "The Influence of Na, K-pump Blockade on Doxorubicin-mediated Cytotoxicity and DNA Strand Breakage in Human Tumor Cells," Cancer Chemother. and Pharmacol. 26:163-167.
While others have noted that normal intracellular potassium stores are essential for lymphocyte activation (Owens, T. and Kaplan, J. G. (1981), "Monovalent Cation Fluxes in Activated Mouse T- and B-Lymphocytes," in Mechanisms of Lymphocyte Activation (Resch, K. and Kirchner, H. eds.) pp. 238-241; Grinstein, S. and Dixon, S. J. (1989), "Ion Transport, Membrane Potential, and Cytoplasmic pH in Lymphocytes: Changes during Activation," Physiol. Rev. 69:417-481; Cahalan, M. D. et al. (1991), "Potassium Channels in Development, Activation, and Disease in T Lymphocytes," in Developmental Biology of Membrane Transport Systems (Benos, D. J., ed.), pp. 357-394), there has been no recent effort to activate immunity through potassium repletion. In the 1940's Dr. Max Gerson devised a nutritional treatment for cancer that included oral administration of various potassium salts. Although he claimed some success, the medical society of New York declared his method of no value. Gerson's therapy attained some fame when he treated the young son of John Gunther for a brain tumor. The boy died. Gerson's therapy is described in his book, A Cancer Therapy: Results of Fifty Cases, (New York: Dura Books, Inc., 1958), pp. 237-248. Although he is deceased, patients are still treated with revised versions of his therapy in Mexico.
The phenomenon of faulty apoptosis or cell suicide has also been implicated in cancer etiology. (Edgington, S. M. (1993), "Looking Death in the Eye: Apoptosis and Cancer Research--Is Cancer really Caused by Cells Refusing to Commit Suicide on Cue?," Bio/Tech. 11:787-792; Hardin, J. A., et al. (1992), "A Simple Fluorescence Method for Surface Antigen Phenotyping of Lymphocytes Undergoing DNA Fragmentation," J. Immuno. Meth. 154:99-107.) It has been suggested that potassium has a role in triggering apoptosis in cells (Ojcius, D. M., et al. (1991), "Ionophore-Induced Apoptosis: Role of DNA Fragmentation and Calcium Fluxes," Exp. Cell Res. 197:43-49).
Thyroid-suppressing drugs and thyroid removal have been shown to increase the virulence of all categories of cancers, carcinogen-induced, transplanted and spontaneous. Statistics relate cancer inversely to thyroid function, with the disease being most common among hypothyroid people and least frequent in hyperthyroids. In one study, breast cancer was nine times as common among extremely hypothyroid and sixteen times as common among thyroidectomized women as among hyperthyroid women. In the early 1950's, Dr. Alfred A. Loeser administered thyroid to breast cancer patients with good results. Others found virtually no response. (McGrady, P. (1964), The Savage Cell, Basic Books, Inc., New York, pp. 138-140.)
It has been known for years that insulin lowers serum potassium concentration. (Kassirer, Jerome P., et al. (1989), in Repairing Body Fluids: Principles and Practice, p. 47). And, it is established that insulin, like many other hormones, neurotransmitters and autacoids, participates in the regulation of immune responses, particularly T-lymphocyte function (Koffler, Michael, et al. (1991), "Immunobiological Consequence of Regulation of Insulin Receptor on Alloactivated Lymphocytes in Normal and Obese Subjects," Diabetes 40:364-370; Coffey, Ronald G. and Hadden, John W. (1984), "Cyclic Nucleotides in Neurohumoral and Hormonal Regulation of Cells of the Immune System," in Stress, Immunity and Aging (Cooper, E. L., ed.) pp. 231 ff.).
When it was discovered that lymphocytes present insulin receptors late in their cycle, investigators studied the effect of the hormone on these leukocytes in vitro. Their studies showed that physiological concentrations of insulin enhance the ability of cytotoxic lymphocytes to injure target cells. Since insulin seemed to have much the same immune enhancing effect as cholinergic agents and to function in the same time and dosage-dependent way, it was hypothesized they worked through the same receptor. However, studies with the muscarinic antagonist, atropine, established that the insulin-induced augmentation of lymphocyte-mediated cytotoxicity (LMC) was independent of the cholinergic receptor (Strom, Terry B., et al. (1975), "Insulin-Induced Augmentation of Lymphocyte-Mediated Cytotoxicity," Science 187:1206-1208). Furthermore, while cholinergic agents cause an increase in cyclic GMP, one investigator was unable to demonstrate any effect of insulin on cytosolic cyclic GMP or AMP, before or after acquisition of its insulin receptor (Ercolani, L., et al. (1985), "Insulin-Induced Desensitization at the Receptor and Postreceptor Level in Mitogen-Activated Human T-lymphocytes," Diabetes: 34:931-937).
It was discovered that lymphocytes could regulate the number of insulin receptors on their membranes. Stimulation by insulin declined as the receptors decreased. Furthermore, an inverse relationship was shown to exist between the concentration of plasma insulin and the ultimate display of receptors. Studies employing the euglycemic clamp technique showed that chronic hyperinsulinemia in vivo caused the number of receptors on cytotoxic T-lymphocytes to decline from 6752 to 1665 per cell, with a corresponding loss of killing effectiveness. Furthermore this loss of cytotoxicity continued in vitro for several hours, despite being placed in low insulin media (Koffler, Michael, et al. (1991), "Immunobiological Consequence of Regulation of Insulin Receptor on Alloactivated Lymphocytes in Normal and Obese Subjects," Diabetes 40:364-370).
A number of investigators studied the effects of insulin on cachexia in tumor-bearing animals. In general they found that it reversed the anorexia and produced a weight gain but did not prolong survival or have any effect on the tumor (Morrison, S. D. (1982), "Feeding Response of Tumor-bearing Rats to Insulin and Insulin Withdrawal and the Contribution of Autonomous Tumor Drain to Cachectic Depletion," Cancer Res. 42:3642-3647; Moley, J. F., et al (1985), "Insulin Reversal of Cancer Cachexia in Rats," Cancer Res. 45:4925-4931; Moley, J. F., et al. (1983), "Effects of Exogenous Insulin Administration on Food Intake, Body Weight Change, and Tumor Doubling Time," Surgical Forum 35:91-93). Schein, Philip S., et al. (1979), "Cachexia of Malignancy," Cancer 43:2070-2076, reported that patients manifesting cachexia of malignancy showed a marked resistance to administered insulin while insulin receptors on monocytes were normal.
Almost thirty years ago, Dr. Oscar Neufeld found similarities between the cachexia of cancer and the wasting in diabetes, and he decided to give insulin to seven patients (three who had cancers of the lung, two of the esophagus, and one each of the liver and stomach). His aim was to stimulate their appetites. In all but one (the stomach-cancer patient), appetite returned; and all of them gained weight and felt better; some experienced a feeling of euphoria. The gradually-increased insulin doses were generally well tolerated. (McGrady, P. (1964), The Savage Cell, Basic Books, Inc., New York, pp. 138-140.)
In 1958, some British, Canadian and American scientists found that insulin had no therapeutic effect on mouse and rat transplanted tumors, although a combination of insulin and glucagon in toxic doses slowed down many of these cancers. Glucagon alone also arrested these cancers. (McGrady, P. (1964), The Savage Cell, Basic Books, Inc., New York, pp. 138-140.)
An increase in cyclic GMP is thought to act as an early signal controlling lymphocyte transformation and proliferation of resting lymphocytes. Acetylcholine and muscarinic cholinergic agents such as pilocarpine are known to raise intracellular levels of cyclic GMP in human lymphocytes. Cholinergic agents also enhance lymphocyte mediated cytotoxicity (LMC) in sensitized cells. Strom, T. B., et al. (1977), "The Role of Cyclic Nucleotides in Lymphocyte Activation and Function," in Progress in Clinical Immunology (Schwartz, R. S., ed.) at 115-153. Acetylcholine and agonists stimulate the immunologic secretion of beta-glucuronidase and enhance phagocytosis in neutrophils. Ignarro, L. J. and Cech, S. Y. (1976), "Bidirectional Regulation of Lysosomal Enzyme Secretion and Phagocytosis in Human Neutrophils by Guanosine 3',5'-Monophosphate and Adenosine 3',5'-Monophosphate (39232)," Proc. Nat. Acad. Sci. 151:448-452. It has also been shown that cholinergic effects are exerted on hematopoietic stem cells. (Unanue, E. R. and Schreiner, G. F. (1975), "The Modulation of Immunoglobulin in B Lymphocytes and its Relevance to Immune Stimulation," in Immune Regulation (Rosenthal, A. S., ed.) at 271.)
Nontoxic treatments for enhancing immune response have been sought, but to date an effective treatment has not been available.